U.S. patent number 6,660,480 [Application Number 10/090,955] was granted by the patent office on 2003-12-09 for method for analyzing nucleic acids by means of a substrate having a microchannel structure containing immobilized nucleic acid probes.
This patent grant is currently assigned to UT-Battelle, LLC. Invention is credited to Robert S. Foote, J. Michael Ramsey.
United States Patent |
6,660,480 |
Ramsey , et al. |
December 9, 2003 |
Method for analyzing nucleic acids by means of a substrate having a
microchannel structure containing immobilized nucleic acid
probes
Abstract
A method and apparatus for analyzing nucleic acids includes
immobilizing nucleic probes at specific sites within a microchannel
structure and moving target nucleic acids into proximity to the
probes in order to allow hybridization and fluorescence detection
of specific target sequences.
Inventors: |
Ramsey; J. Michael (Knoxville,
TN), Foote; Robert S. (Oak Ridge, TN) |
Assignee: |
UT-Battelle, LLC (Oak Ridge,
TN)
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Family
ID: |
25303613 |
Appl.
No.: |
10/090,955 |
Filed: |
March 5, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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460316 |
Dec 14, 1999 |
6376181 |
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848553 |
Apr 28, 1997 |
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Current U.S.
Class: |
435/6.19;
435/285.2; 435/287.2; 436/94 |
Current CPC
Class: |
B01J
19/0093 (20130101); B01L 3/5027 (20130101); B01J
2219/00831 (20130101); B01L 2300/0636 (20130101); B01L
2300/0816 (20130101); B01L 2300/0864 (20130101); B01L
2300/0867 (20130101); B01L 2400/0415 (20130101); B01L
2400/0418 (20130101); B01L 2400/0421 (20130101); B01L
2400/0487 (20130101); Y10T 436/143333 (20150115) |
Current International
Class: |
B01L
3/00 (20060101); B01J 19/00 (20060101); C12Q
001/68 (); C12M 001/34 () |
Field of
Search: |
;435/6,285.2,287.2
;436/94 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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0356160 |
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Feb 1990 |
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EP |
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0620432 |
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Apr 1993 |
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EP |
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2191110 |
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Dec 1987 |
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GB |
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WO 94/05414 |
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Mar 1994 |
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WO |
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WO 95/12808 |
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May 1997 |
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WO |
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Other References
C Effenhauser et al., "Glass Chips for High Speed Capillary
Electrophoresis Separations with Submicrometer Plate Heights",
Anal. Chem., 65:2637-2642 (1993). .
D. Jed Harrison et al., "Capillary Electrophoresis and Sample
Injection Systems Integrated on a Planar Glass Chip", Anal. Chem.,
64:1926-1932 (1992). .
M. Deml et al., "Electric Sample Splitter for Capillary Zone
Electrophoresis", Journal of Chromatography, 320:159-165 (1985).
.
D. Jed Harrison et al., "Micromachining a Minaturized Capillary
Electrophoresis-Based Chemical Analysis System on a Chip", Science,
261:895-897 (1993). .
Dasgupta et al., Anal. Chem, 66:1792-98 (Jun. 1994). .
Wilding et al., Clin. Chem. 40(1), 43-47 (1994). .
Wilidng et al., Clin. Chem. 40(9), 1815-1818 (1994)..
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Primary Examiner: Horlick; Kenneth R.
Attorney, Agent or Firm: Dann Dorfman Herrell and Skillman,
P.C.
Government Interests
This invention was made with government support under Contract No.
DE-AC05-840R21400 awarded by the U.S. Department of Energy to
Lockheed Martin Energy Systems, Inc. and the government has certain
rights in this invention.
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation of U.S. patent application Ser.
No. 09/460,316, filed Dec. 14, 1999, U.S. Pat. No. 6,376,181 which
is a continuation of U.S. patent application Ser. No. 08/848,553,
filed Apr. 28, 1997, now abandoned.
Claims
What is claimed is:
1. A method for analyzing nucleic acids comprising the steps of: a.
providing a substrate having a microchannel structure, at least one
nucleic acid probe being affixed to at least one portion of said
microchannel structure; b. electrokinetically transporting an
analyte nucleic acid to said at least one probe; and, c. analyzing
for the presence of said analyte nucleic acid hybridized to said at
least one probe.
2. A method according to claim 1, wherein the step of
electrokinetically transporting an analyte nucleic acid includes
electroosmotically transporting a solution containing said analyte
nucleic acid through said microchannel structure.
3. A method according to claim 1, wherein the step of
electrokinetically transporting an analyte nucleic acid includes
electrophoretically migrating said analyte nucleic acid through a
solution contained in said microchannel structure.
4. A method according to claim 1, wherein said microchannel
structure is in fluid communication with at least two fluid
reservoirs and the step of electrokinetically transporting an
analyte nucleic acid includes applying electrical voltages to said
at least two fluid reservoirs, one of said reservoirs containing a
solution of said analyte nucleic acid.
5. A method according to claim 1, wherein the step of analyzing for
the presence of said analyte nucleic acid hybridized to said at
least one probe includes electrokinetically transporting a
flourescent label to the probes and detecting fluorescence emission
from said label.
6. A method according to claim 4, wherein said fluorescent label is
an intercalating dye.
7. A method according to claim 1, wherein said analyte nucleic acid
is fluorescently labeled and the step of analyzing for the presence
of said analyte nucleic acid hybridized to said at least one probe
comprises detecting fluorescence emission from said label.
8. A method according to claim 1, additionally comprising the step
of washing said at least one probe following the transport of
analyte nucleic acids to said at least one probe.
9. A method according to claim 4, additionally comprising the step
of washing said at least one probe following the transport of said
fluorescent label to said at least one probe.
10. A method according to claim 8, wherein said washing step
comprises electrokinetically transporting buffer through the
microchannel containing said at least one probe.
11. A method according to claim 9, wherein said washing step
comprises electrokinetically transporting buffer through the
microchannel containing said at least one probe.
12. An apparatus for analyzing nucleic acid comprising: a. a
substrate with a microchannel structure formed therein, said
microchannel structure having a probe channel and at least two end
portions; b. at least one nucleic acid probe affixed within said
probe channel intermediate said two end portions of said
microchannel structure; c. a cover plate affixed to the substrate,
said cover plate and said substrate cooperating to enclose said at
least one nucleic acid probe; d. at least two fluid reservoirs in
fluid communication with said end portions of said microchannel
structure, one of said reservoirs adapted to contain a fluid
comprising at least one substance from the group consisting of
analyte nucleic acid and fluorescent label and, optionally, a
buffer; e. a source of electrical voltage applied to said
reservoirs for effecting electrokinetic transport of said at least
one fluid from said one reservoir through said microchannel
structure to a probe site adjacent to said at least one probe in
said probe channel; and f. a detector for detecting the presence of
said at least one fluid at said probe site.
13. An apparatus according to claim 12, wherein said at least one
fluid comprises an intercalating fluorescent dye, and said detector
is operable to detect fluorescence emission from said dye.
14. An apparatus according to claim 12, wherein said at least two
fluid reservoirs include a first reservoir containing a solution of
said analyte nucleic acid and a second reservoir containing a
solution of a fluorescent label.
15. An apparatus according to claim 14, wherein one end portion of
said microchannel structure is connected to both said first
reservoir and said second reservoir, said voltage source effecting
transport of said fluids to said at least one nucleic acid probe in
said probe channel.
16. An apparatus according to claim 14, wherein said at least two
fluid reservoirs further include a third reservoir containing a
wash buffer, one end portion of said microchannel structure being
connected to said first, said second and said third reservoirs,
said voltage source effecting transport of said fluids to said at
least one nucleic acid probe in said probe channel, and transport
of said wash buffer through said probe channel and past said at
least one nucleic acid probe.
17. An apparatus according to claim 14, wherein said at least two
fluid reservoirs further include a third reservoir containing a
wash buffer, one end portion of said microchannel structure being
connected to said first and said second reservoirs, the other end
portion of said microchannel structure being connected to said
third reservoir, said at least two fluid reservoirs further
including at least two waste reservoirs, said waste reservoirs
being connected to said probe channel at said opposite end
portions, said voltage source, effecting transport of said analyte
nucleic acid and said fluorescent label to said at least one
nucleic acid probe in one direction in said probe channel, and
transport of said wash buffer in the opposite direction through
said probe channel and past said at least one nucleic acid
probe.
18. An apparatus according to claim 12, wherein said at least two
fluid reservoirs include a first reservoir containing a solution of
said analyte nucleic acid, a second reservoir containing a solution
of a fluorescent label, and at least one additional waste
reservoir.
19. An apparatus according to claim 18, wherein one end portion of
said microchannel structure is connected to both said first
reservoir and said second reservoir, and another end portion of
said microchannel structure is connected to said at least one
additional waste reservoir, said voltage source effecting transport
of said analyte nucleic acid and said fluorescent label through
said probe channel and into said at least one additional waste
reservoir.
20. An apparatus according to claim 12, wherein said at least two
fluid reservoirs include a first reservoir containing a solution of
said analyte nucleic acid, a second reservoir containing a solution
of an intercalating dye providing a fluorescent label, and at least
one additional waste reservoir, one end portion of said
microchannel structure being connected to both said first reservoir
and said second reservoir, and another end portion of said
microchannel structure being connected to said at least one
additional waste reservoir, said voltage source applying an
electrical potential between said second reservoir and said at
least one additional reservoir, effecting transport of said analyte
nucleic acid and said dye through said probe channel and into said
at least one additional waste reservoir.
21. An apparatus according to claim 20, wherein said at least one
additional waste reservoir comprises an analyte waste reservoir and
a separate dye waste reservoir.
22. An apparatus according to claim 20, wherein said at least one
additional waste reservoir comprises a combined analyte and dye
waste reservoir.
23. An apparatus according to claim 12, wherein said at least two
fluid reservoirs include a first reservoir containing a solution of
said analyte nucleic acid, a second reservoir containing a wash
buffer, and at least one additional waste reservoir, one end
portion of said microchannel structure being connected to both said
first reservoir and said second reservoir, and another end portion
of said microchannel structure being connected to said at least one
additional waste reservoir, said voltage source applying an
electrical potential between said second reservoir and said at
least one additional reservoir, effecting transport of said analyte
nucleic acid and said wash buffer through said probe channel and
into said at least one additional waste reservoir.
24. An apparatus according to claim 23, wherein said at least one
additional waste reservoir comprises an analyte nucleic acid waste
reservoir and a separate wash buffer waste reservoir.
25. An apparatus according to claim 23, wherein said at least one
additional waste reservoir comprises a combined analyte nucleic
acid and wash buffer waste reservoir.
26. An apparatus for analyzing nucleic acids comprising: a. a
substrate having a microchannel structure formed therein said
microchannel structure including at least two end portions and a
number of different nucleic acid probes being immobilized at
multiple probe sites within said microchannel structure, each of
said different nucleic acid probes being immobilized at a discrete
probe site; b. a cover plate affixed to the substrate, said cover
plate and said substrate cooperating to enclose said probe sites;
c. at least two fluid reservoirs in fluid communication with said
end portions of said microchannel structure, one of said reservoirs
adapted to contain a fluid comprising at least one substance from
the group consisting of analyte nucleic acid and fluorescent label
and, optionally, a buffer; d. a source of electrical voltage
applied to said reservoirs for effecting electrokinetic transport
of said at least one fluid from said one reservoir through said
microchannel structure to a nucleic acid probe site; and e. a
detector for detecting the presence of said at least one fluid at
one of said nucleic acid probe sites.
Description
BACKGROUND OF THE INVENTION
1. Field of The Invention
The present invention relates generally to medical and/or
biological testing and devices for performing same, and more
particularly, to a method and apparatus for analyzing minute
amounts of nucleic acids for the presence of specific nucleotide
sequences. Single-strand DNA probes are bound to specific regions
of microchannels in a glass microchip device. Sub-microliter
volumes of nucleic acid solutions, buffers and other reagents are
transported through the channels under electrokinetic or hydraulic
control. Hybridization of target nucleic acid sequences to
complementary probes is detected using either fluorescent labels or
intercalating fluorescent dyes.
2. Description of the Related Art
Hybridization analysis is typically performed in microtiter plate
wells or on planar surfaces that contain arrays of DNA probes.
Chemical manipulations are required to bring about a hybridization
test and to detect the results. These manipulations presently
include washing or dipping planar arrays into the appropriate
chemicals.
The aforementioned procedures suffer from many drawbacks. For
example, they are wasteful of expensive reagents and limited sample
volumes. Moreover, they are generally not compatible with efficient
automation strategies and thus tend to be time consuming.
A continuing need exists for methods and apparatuses that limit the
use of expensive reagents and priceless samples, while simplifying
the overall procedures to require smaller samples and fewer
processing steps.
SUMMARY OF THE INVENTION
An object of the present invention is to provide a method and
apparatus for analyzing nucleic acids which simplifies chemical
manipulations required to bring about a hybridization test when
performing DNA diagnostics in biomedical, forensic, and research
applications.
Another object of the present invention is to provide a method and
apparatus for analyzing nucleic acids which minimizes the use of
expensive reagents and limited sample volumes.
Another object of the present invention is to provide a method and
apparatus for analyzing nucleic acids which avoids the necessity of
pre-labeling a target DNA and increases the sensitivity of hybrid
detection by reducing background fluorescence due to non-specific
surface adsorption of labeled target DNA.
Still another object of the present invention is to provide a
method and apparatus for analyzing nucleic acids which
significantly extend the usefulness of hybridization diagnostics by
allowing its application to much smaller samples and facilitating
automated processing.
These and other objects are met by providing an apparatus for
analyzing nucleic acids which includes a microchip having a
microchannel structure formed therein, at least one portion of the
microchannel structure having at least one site capable of affixing
thereto a probe, and a plurality of reservoirs in communication
with the microchannel structure for introducing at least one of, or
a mixture of, a reagent, analyte solution, and buffer.
In another aspect of the invention, a method of analyzing nucleic
acids includes bonding oligonucleotide probes to a microchannel
formed in a microchip, adding target nucleic acids and fluorescent
stains to the microchannel, and detecting hybridization by
fluorescence staining of double-stranded DNA.
These together with other objects and advantages which will be
subsequently apparent, reside in the details of construction and
operation as more fully hereinafter described and claimed, with
reference being had to the accompanying drawings forming a part
hereof, wherein like numerals refer to like elements
throughout.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic view of an apparatus for analyzing nucleic
acids according to a preferred embodiment of the present
invention;
FIGS. 2 and 3 are schematic views of different arrangements of
nucleic acid hybridization probes in microchannels;
FIG. 4 is a schematic view of a microchip and microchannel
structure according to another preferred embodiment of the present
invention;
FIG. 5 is a schematic view of a microchip of the present
invention;
FIG. 6 is a photomicrograph showing discrimination of target and
non-target DNA at the intersection of microchannels in the inset
area of FIG. 5 after dsDNA staining with fluorescent dye;
FIG. 7 is a schematic view of another apparatus for analyzing
nucleic acids according to a preferred embodiment of the present
invention; and
FIG. 8 shows fluorescence image profiles of two probe channels
after ds-DNA staining with fluorescent dye.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Referring to FIG. 1, a microchip 16 includes a glass substrate 18
and a cover plate 20 which covers a microchannel structure 22
formed in the upper surface of the substrate 16. The cover plate 20
is permanently bonded to the substrate 18. Both the substrate 18
and cover plate 20 are preferably made of clear glass, and the
substrate may preferably be made from a standard microscope slide.
Alternative construction materials could include plastics (such as
polypropylene, polycarbonate, or polymethylmethacrylate), silicon,
or sapphire.
The microchannel structure 22 is formed using standard
photolithographic techniques, and includes a longitudinal
microchannel manifold portion 24, a first transverse microchannel
portion 26 forming an intersection 28 with the longitudinal portion
24, and a second transverse microchannel portion 30 forming an
intersection 32 with the longitudinal portion 24.
First and second reservoirs 34 and 36 are in fluid communication
with opposite ends of the longitudinal portion 24. The opposite
ends act as ports to introduce the contents of the reservoirs 34
and 36 into the microchannel structure 22. Each reservoir can be a
cylindrical container open at its opposite axial ends, with the
ends of the longitudinal portion 24 being in fluid communication
with the bottom of the container.
Third and fourth reservoirs 38 and 40 are in fluid communication
with opposite ends of the first transverse portion 26. The opposite
ends act as ports to introduce the contents of the reservoirs 38
and 40 into the microchannel structure 22. Each reservoir 38 and 40
is similar in construction to the other reservoirs, with the ends
of the first transverse portion being in fluid communication with
the bottom of each respective reservoir 38 and 40.
Fifth and sixth reservoirs 42 and 44 are in fluid communication
with opposite ends of the second transverse portion 30. The
opposite ends act as ports to introduce the contents of the
reservoirs 42 and 44 into the microchannel structure 22. Each
reservoir 42 and 44 is similar in construction to the other
reservoirs, with the ends of the second transverse portion being in
fluid communication with the bottom of each respective reservoir 42
and 44.
One or more types of single-stranded DNA probes 46 are attached at
individual sites within the microchannel portion 24 of the
microchannel structure 22. The design and fabrication of microchips
and the electrokinetic transport of fluids through the
microchannels is described in U.S. Ser. No. 08/283,769, filed Aug.
1, 1994, hereby incorporated by reference. The microchips described
therein include planar, glass substrates into which the
microchannels are etched photolithographically. The reservoirs
typically hold analyte solutions, buffers, reagents, etc. Typical
microchannel dimensions are 10 .mu.m by 50 .mu.m
(depth.times.width), although channel widths of 1 .mu.m to >100
.mu.m and channel depths of <1 .mu.m to >100 .mu.m may be
used. Voltages are applied to solutions as described in the
aforementioned application to produce electroosmotic flow of fluids
or electrophoretic migration of charged species through the
channels. Alternatively, pressure (or vacuum) may be applied to one
or more fluid reservoirs to cause reagent flow through the
channels.
The individual DNA probes may be arranged in a linear pattern, as
shown in FIG. 2. An alternative embodiment is shown in FIG. 3,
wherein the 46' are arranged in a two-dimensional array in a
widened area 48 of the channel portion 24'. Fluid flow is in the
direction indicated by arrows.
Typically, oligonucleotide probes ten to thirty nucleotides long
are used for hybridization analysis, although much longer probes,
such as DNA restriction fragments or cDNA sequences of >100
nucleotide length, may be used in certain applications.
Oligonucleotide probes may be immobilized by covalent chemical
linkage to the surface. In general, such linkage involves
derivatization of the glass surface with a silane coupling agent,
such as 3-aminopropyltriethoxysilane or
3-glycidoxypropyltrimethoxysilane. An oligonucleotide probe bearing
an alkylamine group at the 5' or 3' end may then be linked to the
surface by direct reaction of its terminal amine with a silane
epoxy group or by cross linking the silane and oligonucleotide
amines using glutaraldehyde or other amine-reactive bi-functional
compounds.
Other immobilization method may also be used. For example,
surface-immobilized avidin or streptavidin may be used to bind
biotinylated probes. Non-covalently adsorbed oligonucleotides on
glass surfaces have also been shown to hybridize to target
sequences.
In the preferred fabrication method, the probes are attached to the
open microchip channels and the cover plate is then bound to the
substrate by a low temperature technique which does not damage the
biomolecules. Such a low temperature bonding technique is described
in application Ser. No. 08/645,497, abandoned, entitled "Low
Temperature Material Bonding Technique" by J. M. Ramsey, R. S.
Foote, and H. Wang, which is incorporated herein by reference.
Individual probes may be applied to specific sites in the channels
by micro-pipeting or other means, such as ink-jet printing. The
separation of individual probes may be facilitated by preparing the
surface with a pattern of reactive, hydrophilic sites separated by
non-reactive, hydrophobic areas. For example, the glass surface may
be treated with an alkyltrialkoxysilane to produce a non-reactive,
hydrophobic surface. Photolithography and chemical etching or laser
ablation may be used to remove the silane layer and expose the
glass substrate in a pattern of separated spots. These spots may
then be treated with a silane coupling agent as described above to
produce reactive, hydrophilic spots. An aqueous probe solution
applied to an individual spot would be confined to its hydrophilic
site and thus prevented from mixing with different probe solutions
in adjacent spots. The intervening hydrophobic regions would also
prevent probe mixing in the case of the other immobilization
methods described above.
Alternatively, the probes may be attached to specific sites in the
channels after standard high-temperature cover plate bonding. Three
methods of achieving this are provided as examples:
(1) The functional group of the silane linker (e.g., the amino
function of 3-aminopropylsilane)may be blocked with a photolabile
protective group. The silane linkers are then de-protected at
specific positions in the channel by exposure to light through the
cover plate using a photolithographic mask or focused beam. Cross
linkers and probes passed through the channel would react only at
de-protected sites. A series of separate de-protection and addition
steps are used to attach a number of different probes to individual
sites.
(2) An array of oligonucleotide probes may be photochemically
synthesized in situ in a parallel fashion.
(3) A channel manifold may be designed to allow the addition of an
individual probe to a given branch or segment of the manifold by
controlling fluid flows.
In the preferred methodology, nucleic acids, buffers and dyes are
electrokinetically driven through the microchannels containing the
immobilized probes. For example, the following sequence of
operations can be used with the device schematically illustrated in
FIG. 4. As seen in FIG. 4, a microchip 50 includes a microchannel
structure 52 connected to a nucleic acid sample reservoir 54, a
buffer reservoir 56, a dye reservoir 58, dye buffer reservoir 60,
and waste reservoir 62. A hybridization chamber 64 is disposed in
the microchannel structure 52 between first and second transverse
portions 66, 68 of the microchannel structure.
A voltage is applied between reservoir 54 which contains the
nucleic acid sample being analyzed and reservoir 56 containing
nucleic acid buffer. For buffers containing a high NaCl
concentration (desirable for rapid nucleic hybridization) the
polarity of reservoir 56 is positive relative to reservoir 54 and
the negatively charged nucleic acids electrophoretically migrate
from reservoir 54 to reservoir 56, passing through the
hybridization chamber 64. Alternatively, a nucleic acid solution
containing a low salt concentration may be electroosmotically
transported into the hybridization chamber by applying a positive
voltage at reservoir 54 relative to reservoir 56. Because
electroosmotic flow toward reservoir 56 is high relative to
electrophoretic migration toward the positive electrode, the net
movement of nucleic acids will be toward reservoir 56 in the later
case. The use of electroosmotic flow versus electrophoretic
migration will depend on a number of factors, and may vary
depending on the type of sample being analyzed. The term
"electrokinetic transport" includes both electroosmotic flow and
electrophoretic migration.
After the DNA sample reaches equilibrium over the probe sites, the
voltage may be discontinued while hybridization occurs. A
double-strand-DNA-specific (dsDNA-specific) fluorescent dye is then
electrokinetically transported through the hybridization chamber 64
by applying voltages to fluid reservoir 58 which contains a dye and
reservoir 60 containing a dye buffer. Because high salt
concentrations are not normally required or desirable for this
step, electroosmotic flow is the preferred method of dye addition
and the polarity of reservoir 58 will normally be positive relative
to reservoir 60. Several fluorescent double-strand-specific nucleic
acid stains are commercially available. Many of these stains are
positively charged so that their electrophoretic migration will be
in the same direction as the electroosmotic flow.
Alternatively, the nucleic acids being analyzed may be pre-labeled
with fluorescent groups by well known procedures. Although this
later method can lead to higher background fluorescence, it may be
preferred in cases where probes contain self-complementary
sequences that can result in stable duplex formation and dye
binding by the probe itself.
Variations in the chip design and analysis procedure are possible.
For example, electrokinetically driven washing steps may be
included before and/or after the dye addition step by applying
appropriate voltages between the buffer reservoirs and a waste
reservoir 62. Nucleic acid and dye solutions might also be added
simultaneously to the hybridization chamber. As an alternative to
electrokinetically driven fluid manipulation, hydraulic pressure or
vacuum may be applied to appropriate reservoirs to control the flow
of solutions through the microchannels.
After completion of the hybridization and dsDNA staining steps, if
used, the hybridization chamber is examined for the presence of
fluorescently labeled sites by illumination with exciting light
through the cover plate. An epifluorescence microscope and CCD
camera may be used, as described below, to obtain a fluorescence
image of the entire chamber or portion thereof. Scanning confocal
fluorescence microscopy may also be used.
The following examples incorporate the apparatus and methodology of
the present invention. Each involves the steps of (1) covalently
bonding oligonucleotide probes to microchannels, (2) adding target
nucleic acids and fluorescent stains to microchannels by
electrokinetic flow, (3) detecting hybridization by fluorescence
staining of double-stranded DNA, and (4) discriminating target and
non-target nucleic acids.
EXAMPLE 1
A 16-mer oligodeoxynucleotide probe sequence containing a
5'-(6-aminohexyl)phosphate [H.sub.2 N--CH.sub.2).sub.6
-5'-pCGGCACCGAGTTTAGC-3'] [SEQ. ID NO: 1] was covalently attached
to the hybridization chamber of a prototype microchip similar to
that shown in FIG. 4 by glutaraldehyde cross linking with the
3-aminopropylsilane-derivatized glass surface. A complementary
16-mer (target sequence) oligodeoxynucleotide in 6.times.SSC buffer
was then electrophoretically added to the hybridization chamber by
applying 0.5 kV between reservoir 56 and reservoir 54 (positive
electrode at reservoir 54) for thirty minutes. A dsDNA-specific
fluorescent dye (TOTO-1, Molecular Probes) in 10 mM Tris-borate
buffer, pH 9.2, was then electroosmotically added to the chamber by
applying 1.0 kV between reservoir 60 and reservoir 58 for 30
minutes. The chip was examined by video microscopy using laser
excitation (514 nm) of fluorescence. Bright fluorescence due to the
dsDNA-bound dye was observed in the hybridization chamber relative
to channels not exposed to the target DNA. The image was recorded
on video tape.
In a subsequent similar experiment using the dsDNA specific dye,
PicoGreen (Molecular Probes), quantification by CCD imaging and
analysis showed a 10-fold increase in fluorescence intensity when
staining was carried out after hybridization of the target DNA,
relative to the intensity observed by staining prior to the
hybridization step.
EXAMPLE 2
The 16-mer oligonucleotide probe of Example 1 was uniformly bound
to the channels of a cross-channel chip shown schematically in FIG.
5 by glutaraldehyde cross-linking. Solutions (50 .mu.M) of the
complementary (target sequence) 16-mer oligodeoxynucleotide (T) and
a non-complementary (non-target sequence) 16-mer
oligodeoxynucleotide (N) in phosphate-buffered saline (PBS) were
then added to separate channels as indicated in FIG. 5, by applying
suction at W for 10 minutes. The channels were then washed with
buffer and dsDNA-specific dye solution (PicoGreen, Molecular
Probes) was added to all channels for five minutes. The
cross-channel intersection was examined by epifluorescence
microscopy using a mercury lamp illumination source and FITC
filters. A 1.0 second CCD exposure, shown in FIG. 6 as the insert
of the broken line area of FIG. 5, showed intense fluorescence
(dark regions) in the channel exposed to target DNA relative to
that of channels exposed to non-target DNA or buffer.
In a similar experiment using laser induced fluorescence imaging,
as described in application Ser. No. 08/800,241, U.S. Pat. No.
6,056,859, entitled "Method and Apparatus for Staining Immobilized
Nucleic Acids" by J. M. and SC Jacobson R. S. Foote, incorporated
herein by reference, signal intensity from channels exposed to
target DNA was 10-fold greater than from channels exposed to
non-target DNA or buffer.
EXAMPLE 3
Two 16-mer probes [H.sub.2 N--(CH.sub.2).sub.6
-5'-GCTAAACTCGGTGCCG-3' (SEQ ID NO: 2) and [H.sub.2
N--(CH.sub.2).sub.6 -5'-pCGGCACCGAGTTTAGC-3' (SEQ ID NO: 2) were
immobilized in separate channels of a cross-channel chip as
indicated in FIG. 7. In FIG. 7, the "T" reservoir is for target
DNA, "B" is for PBS buffer and "W" is for waste.
A solution of 16-mer oligonucleotide (50 nM oligonucleotide in PBS)
complementary to Probe 1 was induced to flow through both channels
for a total of 15 minutes by applying a vacuum at W. The channels
were then washed with buffer and treated with a ds-DNA specific dye
solution (PicoGree, Molecular Probes) for two minutes. After
washing with 10 mM Tris-HCL (pH 8), one mM EDTA (TE) buffer for one
minute, the channels were examined for laser-induced fluorescence
using an argon ion laser at 488 nm and 100 milliwatts power.
Quantitation by CCD imaging, shown in FIG. 8, shows a 4 to 5-fold
greater fluorescence in the Probe 1 channel than in the Probe 2
channel after subtraction of the background signal.
According to the above methods and apparatuses, hybridization
analysis can be performed in a microchip structure that requires
low instrumentation space and extremely low sample/reagent volumes.
The electrokinetic transport of samples and reagents facilitates
automation of sample/reagent manipulations. Moreover, the detection
of hybridization using double-strand DNA-specific fluorescent dyes
eliminates the target DNA labeling step associated with prior art
techniques and increases detection sensitivity.
While the examples referred to above describe nucleic acid probes,
the methodology and apparatuses could also be used for other uses
including, but not limited to, immobilized antibodies for
micro-immunoassays. Numerous biomedical applications can be
envisioned.
While the various embodiments have referred to specific reservoirs
containing specific reagents, buffers or samples, mixtures of two
or more substances can be contained in individual reservoirs. For
example, a reservoir can contain a mixture of reagent and buffer,
buffer and sample, etc.
The many features and advantages of the invention are apparent from
the detailed specification, and thus, it is intended by the
appended claims to cover all such features and advantages of the
invention which fall within the true spirit and scope of the
invention. Further, since numerous modifications and variations
will readily occur to those skilled in the art, it is not desired
to limit the invention to the exact construction and operation
illustrated and described and accordingly, all suitable
modifications and equivalents may be resorted to, falling within
the scope of the invention.
SEQUENCE LISTING <100> GENERAL INFORMATION: <160>
NUMBER OF SEQ ID NOS: 2 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 1 <211> LENGTH: 16 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Sequence source/note="synthetic
oligonucleotide construct containing a 6-amino hexyl phosphate
modification at the 5' end" <400> SEQUENCE: 1 cggcaccgag
tttagc 16 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID
NO 2 <211> LENGTH: 16 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Sequence source/note="synthetic oligonucleotide
construct containing a 6-amino-hexyl phosphate modification at the
5' end" <400> SEQUENCE: 2 gctaaactcg gtgccg 16
* * * * *